Promoters and Control Elements: Designing Expression Cassettes for Gene Therapy

Abstract

It has become apparent that the clinical success anticipated in the field of gene therapy has been limited by progress in several of the fundamental areas of genetics, molecular and cellular biology relevant to its application. Whilst a great deal of effort has been made in the evaluation of transgenes, it is only more recently with the advance of vector systems that attention has begun to be focused upon the means and control of transgene expression. Until recently, the majority of constructs have employed ubiquitous viral promoters to drive expression from simple gene expression cassettes using viral promoters and lacking introns, 3' untranslated regions (UTRs), locus control regions (LCR's), matrix attachment regions (MAR's) and other such genetic components. It has consequently emerged that these elements may have a key role in determining the levels and longevity of gene expression attainable in vivo, irrespective of the vector system utilised. The majority of gene therapy applications would also benefit from the specific optimisation of 'tailormade' expression cassettes to optimise their therapeutic efficacy. In conjunction with modification of vector tropism and strategies to limit their immunogenicity, this should create vectors suitable for the clinical application of gene therapy. This review aims to highlight some of the principle considerations of gene expression in vivo, and the means by which it may most effectively be achieved, whether this is via the minimal modification of an existing eukaryotic promoter or by the more extensive design of a novel promoter and associated elements.

HIV-1-derived lentiviral vectors and fetal route of administration on transgene biodistribution and expression in rhesus monkeys

“…While CMV is a strong viral promoter, several mechanisms have been proposed for transient expression, such as deletion of transduced cells or promoter downregulation. [30][31][32] Even if results of this study suggest that the vector construct containing the CMV promoter was more successful than MND, we cannot conclude that CMV is a more efficient promoter than MND because the quantity of virus injected differed. These results do, however, suggest that the level of transduction is dose-dependent, Figure 6 Whole-tissue fluorescence.…”

Cis-regulatory elements used to control gene expression in plants

“…Consequently, insulators application in multigene constructs is ergonomically beneficial. Insulator sequences guarantee genes autonomy because they determine the compartments in the molecular environment of DNA strands, whereby separating the influence of chromatin state and regulators related to adjacent genes (Papadakis et al 2004;Yang et al 2011).…”

“…They neutralize the position effect, and the influence of adjacent sequences on gene expression (Papadakis et al 2004;Allen 2008), thus protecting genes against undesirable silencing by heterochromatin. Euchromatin domains are separated from heterochromatin by MAR sequences (matrix associated region), known also as SAR (scaffold attachment region), characterized by a high content of AT base pairs.…”

“…The presence of 3′UTR and its content significantly affect the final gene expression (Papadakis et al 2004). This property was confirmed during analysis of β-carotene content.…”

Melanoma-specific expression in first-generation adenoviral vectors in vitro and in vivo — use of the human tyrosinase promoter with human enhancers

“…The transient expression obtained with first-generation Ad vectors is well known, and lately it was found that the major mechanism behind the decreased expression observed was transcriptional silencing due to methylation of the CMV promoter. 21 Silencing is most likely a consequence of the origin of the sequences in the promoter, and even though the over time fate of the 2hE-hTyrP promoter has not been investigated, we hypothesize that silencing of this construct is less likely to occur, as it contains only human sequences (for review see Papadakis et al 22 ).…”